Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution.

Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.     

Modified DNA fragments activate NaeI cleavage of refractory DNA sites.

Endonuclease NaeI cleaves DNA using a two-site mechanism. The DNA-binding sites are nonidentical: they recognize different families of flanking sequences. A unique NaeI site that is resistant to cleavage resides in M13 double-stranded DNA. NaeI can be activated to cleave this site by small DNA fragments containing one or more NaeI sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the NaeI scissile bonds (S-activator) is not cleaved by NaeI, even though this S-activator binds to the substrate site. The S-activator activates NaeI to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that NaeI reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.     

Dissection of functional domains in phage fd adsorption protein. Discrimination between attachment and penetration sites

We constructed a set of deletion mutants in the attachment protein of phage fd. These mutants lack sequences coding for sections in the amino-terminal half. All the mutants that comprise a leader sequence are incorporated into phage particles. Our data strongly suggest a bipartite organization of the amino-terminal domain with (1) a region for receptor recognition and (2) a region that is necessary for penetration of the DNA into the host cell. These regions were mapped. Some evidence suggesting different roles for gene 3 protein in penetration of the outer and inner membrane are discussed. We demonstrate that the phenotypes caused by gene 3 protein in host cells can be subdivided into two groups with different sequence requirements: (1) phenotypes related to outer membrane disturbance; and (2) phenotypes related to the tolQRA transport system.     

Dinucleoside monophosphates. I. Optical properties and conformation in solution with one base charged.

A least squares analysis of the titration properties of several dinucleoside monophosphates enables calculation of the pK's for protonation. These pK's are used to resolve the spectral properties of dinucleoside monophosphates with one base charged from the apparent spectral properties of a dinucleoside monophosphate in aqueous solution. This method is applied to dinucleoside monophosphates containing adenosine and/or cytidine. Results of CD, nmr, and CD-temperature dependence measurements are presented. The results indicate that singly protonated dimers of these nucleosides stack as do their unprotonated analogs. It is suggested that this is true for all dimers with one base charged.     

Physiological bases of genetic differences in cannibalism behavior of the confused flour beetle Tribolium confusum

Physiological causes of genetic differences in cannibalism were examined to gain a better understanding of constraints on behavior evolution. Cannibalism has complex population level consequences in Tribolium confusum, including dramatic effects on population size. Laboratory strains with low and high cannibalism rates, obtained through inbreeding, have maintained distinct levels of cannibalism for over two decades even in the absence of artificial selection to maintain the differences. Why strains differ in their cannibalism rates was examined by measuring: (1) the nutritional benefit from cannibalism in both nutritionally good and poor environments, and (2) the possibility that eggs are an important source of water. How strains achieve differences in cannibalism was examined by testing for differences between strains in their ability to find eggs and in their tendency to eat eggs. Beetles from both strains survive equally well in a nutritionally good environment, but they accomplish this in different ways. The low cannibalism strain has high survivorship with and without cannibalism. The high cannibalism strain has low survivorship when not fed eggs and survivorship equivalent to the low cannibalism strain when fed eggs, suggesting it compensates for poor nutritional adaptation by eating eggs. The strains also differ in feeding behavior; beetles from the high cannibalism strain have a higher appetite for eggs. Beetles from the two strains did not differ in locomotor activity, search efficiency, or need for water. The observed behavioral and nutritional differences may contribute to the maintenance of different levels of cannibalism.     

Structure of NaeI-DNA complex reveals dual-mode DNA recognition and complete dimer rearrangement

NaeI, a novel DNA endonuclease, shows topoisomerase and recombinase activities when a Lys residue is substituted for Leu 43. The NaeI-DNA structure demonstrates that each of the two domains of NaeI recognizes one molecule of DNA duplex. DNA recognition induces dramatic rearrangements: narrowing the binding site of the Topo domain 16 A to grip DNA, widening that of the Endo domain 8 A to encircle and bend DNA 45 degrees for cleavage, and completely rebuilding the homodimer interface. The NaeI-DNA structure presents the first example of novel recognition of two copies of one DNA sequence by two different amino acid sequences and two different structural motifs in one polypeptide.     

Oxidative DNA base damage, antioxidant enzyme activities and selenium status in highly iodine-deficient goitrous children

The objective of this study was to investigate oxidative DNA damage, and the levels of antioxidant enzymes (AOE) and selenium (Se) in relation to iodine deficiency and/or goiter in children. The study was performed in a group of goitrous high school children (15-18 years of age) ( n =14) with severe or moderate iodine deficiency. Thyroid hormones (TSH, FT 4 , TT 4 , FT 3 , TT 3 ), urinary iodine (UI) and plasma Se levels, and erythrocyte glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and catalase (CAT) activities were determined and compared with those of a control group consisting of non-goitrous high school children ( n =14) with normal UI levels or mild iodine deficiency. In the goitrous group, concentrations of FT 4 , TT 4 , plasma Se and UI, and activities of GSHPx and SOD were found to be significantly lower. Six typical hydroxyl radical-induced base lesions in genomic DNA of peripheral blood were identified and quantified by gas chromatography/isotope-dilution mass spectrometry (GC/IDMS), and higher levels of DNA base lesions were observed in the goitrous group. The results suggest that highly iodine-deficient goitrous children may be under oxidative stress, which may lead to greater level of oxidative damage to DNA. This study supports the evidence for the reported relationship between iodine deficiency and the increased incidence of thyroid malignancies.     

Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase

NaeI is a type IIe endonuclease that interacts with two DNA recognition sequences to cleave DNA. One DNA sequence serves as a substrate and the other serves to activate cleavage. NaeI is divided into two domains whose structures parallel the two functionalities recognized in NaeI, endonuclease and topoisomerase. In this study, we report evidence for mutations that break interdomain functional communication in a NaeI-DNA complex. Deletion of the initial 124 amino acids of the N-terminal domain of NaeI converted NaeI to a monomer, consistent with self-association being mediated by the Endo domain. Deletions within a small region of the C-terminal DNA binding domain of NaeI (amino acids 182-192) altered the recognition by NaeI of sequences flanking the NaeI recognition sequence. Substituting Ala for Arg182 within this region had no apparent effect on DNA binding but greatly reduced the extent of DNA cleavage even though it is not part of the catalytic Endo domain. Substituting Ala for Ile185 reduced the extent of DNA binding about 1000-fold. Substituting Ala for Lys189 altered flanking sequence recognition. Residues 182-192 are away from the Endo domain responsible for cleavage and also face away from the modeled DNA binding faces of the apoprotein crystal structure. We propose that residues 182-192 are part of a web that mediates the flow of information between the NaeI Endo and Topo domains.     

Resolution of (±)‐β‐methylphenylethylamine by a novel chiral stationary phase for Pirkle‐type column chromatography

In this study, a new Pirkle‐type chiral column stationary phase for resolution of β‐methylphenylethyl amine was described by using activated Sepharose 4B as a matrix, L ‐tyrosine as a spacer arm, and an aromatic amine derivative of L ‐glutamic acid as a ligand. The binding capacities of the stationary phase were determined at different pH values (pH = 6, 7, and 8) using buffer solutions as mobile phase, and enantiomeric excess (ee) was determined by HPLC equipped with chiral column. The ee was found to be 47%. Chirality, 2010. © 2009 Wiley‐Liss, Inc.